Supercritical CO2 Foaming of Thermoplastic Materials Derived from Maize: Proof-of-Concept Use in Mammalian Cell Culture Applications

Background: Foams are high porosity and low density materials. In nature, they are a common architecture. Some of their relevant technological applications include heat and sound insulation, lightweight materials, and tissue engineering scaffolds. Foams derived from natural polymers are particularly attractive for tissue culture due to their biodegradability and bio-compatibility. Here, the foaming potential of an extensive list of materials was assayed, including slabs elaborated from whole flour, the starch component only, or the protein fraction only of maize seeds. Methodology/Principal Findings We used supercritical CO2 to produce foams from thermoplasticized maize derived materials. Polyethylene-glycol, sorbitol/glycerol, or urea/formamide were used as plasticizers. We report expansion ratios, porosities, average pore sizes, pore morphologies, and pore size distributions for these materials. High porosity foams were obtained from zein thermoplasticized with polyethylene glycol, and from starch thermoplasticized with urea/formamide. Zein foams had a higher porosity than starch foams (88% and 85%, respectively) and a narrower and more evenly distributed pore size. Starch foams exhibited a wider span of pore sizes and a larger average pore size than zein (208.84 vs. 55.43 μm2, respectively). Proof-of-concept cell culture experiments confirmed that mouse fibroblasts (NIH 3T3) and two different prostate cancer cell lines (22RV1, DU145) attached to and proliferated on zein foams. Conclusions/Significance: We conducted screening and proof-of-concept experiments on the fabrication of foams from cereal-based bioplastics. We propose that a key indicator of foamability is the strain at break of the materials to be foamed (as calculated from stress vs. strain rate curves). Zein foams exhibit attractive properties (average pore size, pore size distribution, and porosity) for cell culture applications; we were able to establish and sustain mammalian cell cultures on zein foams for extended time periods

Two different prostate cancer cell lines attach and proliferate on zein foams.

<p>A portion of the porous surface of a wpTPZ foam as observed by confocal microscopy at 20X (A) before cell seeding, and (B) 22RV1 cells after seven days of growth. Prostate cancer cell lines cultured on wpTPZ attach, proliferate, and develop into tree-like structures on the edge of wpTPZ foams: (C) 22RV1 cells observed at the third day of culture (24X; stereoscopic microscope); (D) Du145 cells observed at the third day of culture (24X; stereoscopic microscope).</p

Relevant indicators of the quality of foams obtained by supercritical CO₂ expansion in slabs derived from thermoplasticized starch plasticized with urea/formamide (TPSuf foams) and thermoplasticized zein (TPZ foams).

<p>¹Pore size is expressed as the projected area of the pore as determined by image analysis of electronic microscope micrographs.</p><p>²Higher ratios of Thickness/Surface expansion indicate more spherical pores.</p><p>Relevant indicators of the quality of foams obtained by supercritical CO₂ expansion in slabs derived from thermoplasticized starch plasticized with urea/formamide (TPSuf foams) and thermoplasticized zein (TPZ foams).</p

Scanning electronic microscope (SEM) micrographs of transverse cuts of slabs made from whole flour or starch/zein blends after exposure to CO₂ supercritical foaming:

<p>(A) TPBM120 slab (3000X magnification); (B) TPBM120 slab (6000X magnification); (C) Mix[TPZ&TPSsg] slab (1500X magnification); (D) Mix[TPZ&TPSsg] slab (3000X magnification), (E) Mix[TPZ&TPSuf] slab (3000X magnification), and (F) TPmBM slab (1500X magnification).</p

Maize-derived thermoplastic were produced by (A) thermo-extrusion in a twin conical screw mini extruder (Haake MiniLab, Thermo Scientific, USA) followed by thermopressing at 20 MPa for 4 min in a P300P press (Collin, Germany).

<p>(B) Slabs of these plastics were subjected to supercritical CO₂ foaming, which occurred in two stages: (C) diffusion and solubilization of CO₂ molecules within a solid material matrix at supercritical conditions, and (D) a sudden drop in pressure allows the formation of CO₂ bubbles within the material.</p

Scanning electronic microscope (SEM) micrographs and pore size distributions of foams.

<p>Foams made from (A) starch slabs thermoplasticized at 135°C and 50 rpm using urea/formamide as a plasticizer (sample TPSuf); observed at 1500X, and (B) at 2000X magnification. Foams made from zein slabs thermoplasticized at 75°C and 50 rpm (sample TPZ); observed at (C) 1500X, and (D) 2000X magnification. (E) The cumulative distribution of pore sizes, as calculated by image analysis of SEM micrographs, is presented for TPSuf foams (blue line) and Z foams (yellow line). Pore sizes are expressed in terms of projected areas ([=] μm²). The frequency distribution of pore sizes calculated by image analysis of SEM micrographs is presented for (F) TPSuf foams, and (G) TPZ foams.</p

Schematic representation of the experimental treatments and materials derived from them:

<div><p>TPZ: thermoplasticized zein; TPS: thermoplasticized starch; TPBMx: thermoplasticized blue maize (x is a suffix that indicates extrusion temperature); TPmBM: thermoplasticized chemically modified blue maize (as described in materials and methods); Mix[TPS_y+TPZ]^a: thermoplasticized blends of TPS and TPZ (80:20 wt/wt).</p> <p>The y subindex indicates the plasticizer used to produce TPS. ^aBlends were produced using the close mode compounding described in materials and methods. Plasticizers used where sg (sorbitol-glycerol); uf (urea-formamide); and PEG400 (poly-ethylene glycol with m.w. = 400 Da).</p></div

(A) Samples of maize-derived thermoplastics exposed to supercritical CO₂ foaming exhibited different fold increases in thickness (blue), surface area (yellow), and volume (red).

<p>Insets show photographic images of samples elaborated from TPS, TPZ, and TBM140 (B) before and (C,D) after exposure supercritical CO₂ foaming.</p

Processing conditions used to elaborate maize derived bioplastics later exposed to CO₂ supercritical foaming conditions.

<p><b>Plasticizers:</b> Peg400: polyethylene-glycol 400; uf: urea/formamide; sg: sorbitol and glycerol mixture (1.4:1 wt/wt).</p><p><b>Bioplastics:</b> TPZ: thermoplasticized zein; TPS: thermoplasticized starch; TPBMx: thermoplasticized blue maize (x is a suffix that indicate extrusion temperature); TPmBM: thermoplasticized chemically modified blue maize (as described in materials and methods); Mix[TPS_y+TPZ]^a: thermoplasticized Blends of TPS and TPZ (80:20 wt/wt). The y subindex indicates the plasticizer used to produce TPS. ^aBlends were produced using the close mode compounding described in materials and methods.</p><p>Processing conditions used to elaborate maize derived bioplastics later exposed to CO₂ supercritical foaming conditions.</p

Fibroblasts anchor and proliferate on zein foam surfaces.

<p>Confocal microscopy images showing DAPI and MitoTracker co-stained fibroblasts growing on zein foam surfaces. (A) Confocal image showing the intrinsic fluorescence of a zein foam surface (green fluorescent x-y plane). A line of cells (indicated by a single-head arrow), which developed on the edge of the x-y plane, can be seen (blue cell nuclei and red-stained mitochondria). (B) A confocal 2D cut at a slightly closer x-y plane (above that shown in A) reveals the presence of active cells covering the zein surface. DAPI and MitoTracker staining was used to reveal cell nuclei (blue) and mitochondria of metabolically active cells (red). A thick multi-layer of cells is indicated with a double-headed arrow. Green fluorescent regions, corresponding to the zein foams underneath the layer of cells, can be observed (indicated with green arrows). (C) Fibroblasts seeded on zein foams and fed continuously at 0.003 mL min^-1 in a flow chamber linearly increased their global glucose consumption during the first 60 h of continuous culture. Dotted line indicates best linear fit. Error bars indicate standard deviation of three independent replicates. (D) Scheme of the continuous flow chamber (effective volume of 100 μL).</p